The present invention generally relates to a plant nitrogen regulatory PII gene (hereinafter P-PII gene), a gene involved in regulating plant nitrogen metabolism. The invention provides P-PII nucleotide sequences, expression constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P-PII gene from said constructs. The invention also provides substantially pure P-PII proteins.
The P-PII nucleotide sequences and constructs of the invention may be used to engineer organisms to overexpress wild-type or mutant P-PII regulatory protein. Engineered plants that overexpress or underexpress P-PII regulatory protein may have increased nitrogen assimilation capacity. Engineered organisms may be used to produce P-PII proteins which, in turn, can be used for a variety of purposes including in vitro screening of herbicides. P-PII nucleotide sequences have additional uses as probes for isolating additional genomic clones having the promoters of P-PII gene. P-PII promoters are light- and/or sucrose-inducible and may be advantageously used in genetic engineering of plants.
Plants can assimilate soil ammonia or nitrate reduced to ammonia into organic form in leaves or roots. Ammonia assimilation into glutamine and glutamate occurs primarily in leaf chloroplasts or in root plastids by the combined action of chloroplast glutamine synthetase (GS2; GLN2 gene) and glutamate synthase (GOGAT) (Miflin, B. J. and Lea, P. J., 1977, Ann. Rev. Plant Physiol. 28:299-329). As the assimilation of inorganic nitrogen into organic form requires carbon skeletons, reducing equivalents, and ATP, light serves to coordinate nitrogen assimilation with photosynthesis. Genes involved in plant nitrogen assimilation are induced directly by light (via phytochrome), as well as indirectly by metabolic changes in photosynthate. For example, it has been shown that sucrose supplementation to plant growth media can at least partially induce the expression of mRNA for GLN2 or nitrate reductase (NR) in the absence of light (Cheng et al., 1992, Proc. Natl. Acad. Sci. USA. 89:1861-1864; Faure et al., 1994, Plant J. 5:481-491). Conversely, sucrose can repress the expression of asparagine synthetase (ASN1) (Lam et al., 1994, Plant Physiol. 106:1347-1357). More recently, it has been shown that the effects of sucrose on gene expression can be reversed by the addition of an organic nitrogen source both for nitrate reductase (NR) (Vincentz et al., 1993, Plant J. 3:315-324) and for ASN1 (Lam et al., 1994, Plant Physiol. 106:1347-1357). These findings indicate that plants are able to sense levels of carbon and organic nitrogen, and in turn modulate the expression of genes involved in nitrogen assimilation.
Bacteria can also assimilate ammonia into glutamate or glutamine. Plants"" ability to sense changes in the levels of carbon and nitrogen metabolites is reminiscent of a nitrogen regulatory system (Ntr) in bacteria in which a protein called PII, encoded by the glnB gene, can regulate the assimilation of nitrogen into glutamine via glutamine synthetase (GS; glnA) in response to changes in the ratio of organic nitrogen to carbon metabolites (Magasanik, B., 1994, J. Cell. Biochem. 51:34-40).
In response to changes in the metabolic status (i.e., ratio of glutamine to xcex1-ketoglutarate [gln/xcex1-KG]), the PII protein of bacteria interacts with a set of partners to regulate the glnA gene at the transcriptional level, and to regulate GS enzyme activity at the post-translational level (Magasanik, B., 1994, J. Cell. Biochem. 51:34-40). Changes in the gln/xcex1-KG ratio affect the activity of the PII protein via a post-translational modification (uridylylation) at Tyr51 (Magasanik, B., 1988, TIBS 13:475-479). In response to low gln/xcex1-KG, the PII protein is uridylylated by uridylyltransferase (UTase) (id.). The PII-UMP thus formed then interacts with an adenylyltransferase (ATase) to deadenylylate the GS-AMP enzyme and thereby activate the GS enzyme (Magasanik, B., 1994, J. Cell. Biochem. 51:34-40). A high gln/xcex1-KG ratio causes the deuridylylation of PII-UMP. This unmodified form of PII interacts with ATase to stimulate the adenylylation and inactivation of the GS enzyme. The ability of ATase to attach or remove AMP from the GS enzyme is dependent on the interaction of ATase with PII or PII-UMP, respectively (Foor et al., 1975, Proc. Natl. Acad. Sci. USA 72:4844-4848). Thus, the nitrogen-regulatory protein PII, is a signal transducer whose post-translational modification indirectly regulates GS enzyme activity post-translationally. In addition to its ability to regulate the GS holoenzyme activity, PII can also interact with a two-component system (NRII/NRI, or NtrB/NtrC) to regulate the transcription of the glnA gene encoding GS (Magasanik, B., 1994, J. Cell. Biochem. 51:34-40). Under low gln/xcex1-KG levels, NRII-kinase phosphorylates NRI which then interacts with the "sgr"54 to activate glnA gene expression (Ninfa, A. J. and Magasanik, B., 1986, Proc. Natl. Acad. Sci. USA 83:5909-5913). When the gln/xcex1-KG ratio is high, the interaction of PII with NRII stimulates the NRII-phosphatase activity to dephosphorylate NRI-phosphate, and turn off the inducible promoter of glnA transcription (Ninfa, A. J. and Magasanik, B., 1986, Proc. Natl. Acad. Sci. USA 83:5909-5913). Thus, the nitrogen-regulatory protein PII works in concert with other proteins, including UTase, ATase, NRII, and NRI, to regulate glutamine synthetase enzyme activity or glnA transcription in response to the ratio of organic nitrogen to carbon metabolites (Magasanik, B., 1994 J. Cell. Biochem. 51:34-40).
To date, PII homologues have been identified in a diverse set of bacteria including enteric bacteria (Magasanik, B., 1994, J. Cell. Biochem. 51:34-40), cyanobacteria (Tsinoremas et al., 1991, Proc. Natl. Acad. Sci. USA 88:4565-4569), Bacillus (Wray et al., 1994, J. Bacteriol. 176:108-114), and in archaebacteria (Souillard, N. and Sibold, L., 1989, Mol. Microbiol. 3:541-551).
The present invention relates to a plant nitrogen regulatory P-PII gene involved in regulating nitrogen assimilation in plants. The invention provides P-PII coding nucleotide sequences, expression constructs comprising P-PII coding sequences, and host organisms, including plants, containing said expression constructs. The invention also provide P-PII proteins.
The invention is based on the surprising discovery that plants have a structural homolog, P-PII, to the bacterial PII protein. This is the first time a PII-like gene has been identified in an eukaryote. The regulation of P-II mRNA levels by light and by metabolites, such as sucrose, parallels those of nitrogen assimilatory genes such as chloroplastic GS2 (GLN2). See Faure et al., 1994, Plant J. 5:481-491; Edwards, J. W. and Coruzzi, G. M., 1989, Plant Cell 1:241-248; Lam et al., 1994, Plant Physiol. 106:1347-1357; Vincentz et al., 1993, Plant J. 3:315-324. These findings indicates that like bacterial PII, P-PII protein is a plant nitrogen regulatory protein that controls the expression of nitrogen assimilation functions.
The P-PII nucleotide sequences and constructs of the invention may be advantageously used to engineer plants to overexpress P-PII regulatory protein. P-PII overexpression or underexpression should enhance the levels of certain nitrogen assimilation functions and thereby increase nitrogen utilization efficiencies of engineered plants.
P-PII nucleotide sequences and constructs of the invention also may be used to engineer organisms to overexpress wild-type or mutant P-PII regulatory protein. Full length cDNAs for P-PII can be used in a xe2x80x9creverse biochemicalxe2x80x9d approach to synthesize and characterize the encoded P-PII proteins. The ability to use the cloned P-PII to synthesize the purified P-PII proteins will allow a characterization of P-PII protein in terms of physical properties (i.e., inducer or activator preference) and subcellular localization (i.e., plastid vs. cytosol).
Full length P-PII cDNA clones may also be used to synthesize highly purified preparations of the wild-type or altered P-PII in vitro or in vivo (e.g., bacteria, algae, neurospora, yeast, plant, or animal cells). In vitro or in vivo synthesized P-PII protein can be used as a substrate in a screen to identify novel herbicidal compounds, which selectively inhibit this nitrogen regulatory protein. The isolated cDNAs encoding P-PII may also be used to create plants resistant to such herbicides.
Nucleic acids, DNA or RNA, encoding P-PII have additional uses as probes for isolating additional genomic clones having the promoters of P-PII genes. P-PII promoters are light- /or sucrose-inducible and may be advantageously used in genetic engineering of plants. For example, P-PII promoters may be used to directly express genes encoding functions that P-PII proteins directly or indirectly activate or induce. P-PII promoter sequences also may be used to construct chimeric promoters.
Peptide and polypeptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows:
A (alanine)
R (arginine)
N (asparagine)
D (aspartic acid)
C (cysteine)
Q (glutamine)
E (glutamic acid)
G (glycine)
H (histidine)
I (isoleucine)
L (leucine)
K (lysine)
M (methionine)
F (phenylalanine)
P (proline)
S (serine)
T (threonine)
W (tryptophan)
Y (tyrosine)
V (valine)